Study Of HGPRT Gene Knock-down And Knock-out Medical Model In Rabbits

Compared to mouse, rabbits possess characters of physiology, anatomy and evolution closer to human race. Rabbits have a relatively less breeding cost, making them suitable for studies of the cardiovascular, pulmonary and metabolism diseases. Thus, gene-modified rabbit model should find wide applications and market value in medical research. Recently the technology for somatic cells cloned rabbit has been successfully established, which makes it possible to produce rabbits with modifications to specific genes by the combination of RNA interference or homologous recombination and somatic cell nuclear transfer.This study is dedicated to smooth the way of establishing the HGPRT-knockdown or knockout rabbit model. Our work includes two parts. In the first part, HGPRT-knockdown plasmids were devised and transfected into rabbit fibroblasts. The stable transgenic fibroblast lines were generated, of which the average positive rate was 83.3% defined by PCR. The mRNA and protein levels of HGPRT were markedly lower in transgenic fibroblast lines as compared with that of the control. When the transgenic fibroblasts were used as donor cells of nuclear transfer, the blastocyst rate were 27.8%. Compared with the normal group the difference was considered not significant. In the second part, rabbit full length HGPRT gene BAC clone LBNL1-304M19 was used as the template. A 47kb rabbit HGPRT gene fragment, which does not have promoter and exon1, was cloned into pBACLinkSp plasmid to form pBACLinkSp-rHGPRT recombinant plasmid via Gap-Repair by Red recombination system. Then, different homologous arms were designed to delete different coding region of HGPRT gene on the basis of the pBACLinkSp-rHGPRT plasmid. Three different HGPRT gene targeting vectors was constructed. Meanwhile, the efficiency of deleting different sizes of DNA fragment by homologous recombination technology also was studied.In the present study, our results indicated that the HGPRT mRNA and protein levels in the transgenic fibroblast lines were significantly lower than that in the negative control, the nuclear transfer -embryo was developed to the blastocyst stage and embryo transplantation in progress; then three different HGPRT gene targeting vectors have been constructed by Red recombination system, HGPRT-knockout fibroblast clones were being screened and identified. These experiments form the basis for studying HGPRT-associated diseases, exploring the gene targeting in rabbit fibroblast cells and embryonic stem cells, generating the HGPRT-knockdown or knockout rabbit model.